Using double-labelling immunofluorescent histochemistry, calretinin (CR), a calcium binding protein, was found to coexist with calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), and substance P (SP) but not calbindin-D28K and galanin in the fibers innervating the lamina propria of the rat intestinal villi. An acetylcholinesterase (AChE) histochemical stain revealed that the majority of CR cells in the myenteric ganglia were cholinergic and about one half of the submucosal CR cells contained AChE. In situ hybridization studies confirmed the presence of CR mRNA in the dorsal root ganglia (DRG) had a ribonuclease protection assay verified the presence of CR message in the intestine. The coexistence of CR in CGRP cells of the DRG which also contain SP and VIP suggest that the DRG may be the source of the quadruple colocalization within the sensory fibers of the villi. While the function of CR in these nerves is unknown, it is hypothesized that the coexistence of the 3 potent vasodilatory peptides influence the uptake of metabolized food products within the vasculature of the villi.